Bispecific antibodies (BsAbs), also called bifunctional antibodies, are multivalent antibodies with specific binding sites for two antigenic determinants and which can react with two types of antigens. BsAbs can be produced using hybrid hybridomas, or more specifically, quadromas which are fusions of two different types of monoclonal antibody-producing cells (U.S. Pat. No. 4,474,893; R. Bos and W. Nieuwenhuitzen (1992) Hybridoma 11(1): 41-51). BsAbs can also be generated by linking Fab (antigen-binding) fragments or Fab′ fragments of two types of monoclonal antibodies, using chemical techniques (M. Brennan et al. (1985) Science 229(1708): 81-3) or by genetic engineering. In addition, BsAbs can be produced by covalently linking two complete monoclonal antibodies (B. Karpovsky et al. (1984) J. Exp. Med. 160(6): 1686-701).
Problems underlying BsAb production methods include the possibility of generating ten different types of antibody molecules due to random combination of immunoglobulin heavy chains and light chains (M. R. Suresh et al. (1986) Methods Enzymol. 121: 210-28). Among the ten types of antibodies produced by quadromas, the only antibody that has the desired dual specificity is the one that has the correct light and heavy chain combination and which is composed of two light chain/heavy chain pairs having different binding specificities. Therefore, the antibody having the desired dual specificity must be selectively purified from the ten types of antibodies produced by quadromas. Purification is generally performed using affinity chromatography, but this method is laborious and has low yields (Y. S. Massimo et al. (1997) J. Immunol. Methods 201: 57-66).
Methods that overcome such problems and give higher BsAb yields include, for example, methods of chemically linking antibody fragments such as Fab′-thionitrobenzoic acid derivative and Fab′-thiol (SH) (Brennan et al. (1985) Science 229: 81). Furthermore, methods for more conveniently obtaining chemically linkable Fab′-SH fragments include methods for producing these fragments from hosts such as E. coli using genetic recombination techniques (Shalaby et al. (1992) J. Exp. Med. 175: 217-25). Genetic recombination techniques can also be used to obtain BsAbs composed of humanized antibody fragments. Diabodies (Db) are BsAbs constructed from gene fusion of two types of fragments, and comprise a light chain variable region (VL) connected to a heavy chain variable region (VH) by a linker that is too short to allow pairing between them (P. Holliner et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-8; EP No. 404,097; WO93/11161). An example of such a Db that has been further improved is single-chain Db (Japanese Patent Application No. 2002-112369). However, antibody fragments have a shorter serum half-life when compared to full-length antibodies, and do not have effector functions as complete antibodies do. Therefore, in some cases, full-length antibodies are more suitable for diagnosis and therapy.
Methods for efficiently linking generated antibody heavy chains into heterodimers include the method for introducing a sterically complementary mutation into the CH3 domain (a portion of the constant region) in the multimerized domain of an antibody heavy chain (Ridgway et al. (1996) Protein Eng. 9: 617-21). Heavy chains produced by this method may still form pairs with the wrong light chains. Patent Document 1 describes a method for generating multi-specific antibodies which share common light chains with heteromeric polypeptides having antigen-binding domains, and bind to these polypeptides.
BsAbs having specific binding capacities for two different antigens are useful as targeting agents in clinical fields such as in vitro and in vivo immunodiagnosis, therapy, and immunoassay. For example, they can be used as vehicles to link enzymes to carriers by designing a BsAb so that one of its arms binds to an epitope of an enzyme reaction non-inhibiting portion of an enzyme to be used in an enzyme immunoassay, and the other arm binds to a carrier for immobilization (Hammerling et al. (1968) J. Exp. Med. 128: 1461-73). Another example is antibody-targeted thrombolytic therapy. This therapy examines the use of antibodies that transport enzymes such as urokinase, streptokinase, tissue plasminogen activator, prourokinase, and such, and their precursor proteins, in a manner specific to fibrin in thrombi (T. Kurokawa et al. (1989) Bio/Technology 7: 1163; Unexamined Published Japanese Patent Application No. (JP-A) Hei5-304992). Furthermore, uses of BsAbs have been reported as potential mouse/human-chimeric bispecific antibodies for cancer targeting (JP-A Hei2-145187), and in cancer therapy and diagnosis for various tumors (see for example, JP-A Hei5-213775; JP-A Hei10-165184; JP-A Hei11-71288; Published Japanese Translation of International Publication No. 2002-518041; Published Japanese Translation of International Publication No. Hei11-506310; Link et al. (1993) Blood 81: 3343; T. Nitta et al. (1990) Lancet 335: 368-71; L. deLeij et al. (1990) Foundation Nationale de Transfusion Sanguine, Les Ulis France 249-53; Le Doussal et al. (1993) J. Nucl. Med. 34: 1662-71; Stickney et dl. (1991) Cancer Res. 51: 6650-5), mycotic therapy (JP-A Hei5-199894), immune response induction (Published Japanese Translation of International Publication Hei 10-511085; Weiner et al. (1993) Cancer Res. 53: 94-100), induotion of killer T-cell function (Kroesen et al. (1994) Br. J. Cancer 70: 652-61; Weiner et al. (1994) J. Immunol. 152: 2385), immunoanalysis (M. R. Suresh et al. (1986) Proc. Natl. Acad. Sci. USA 83: 7989-93; JP-A Hei5-184383), immunohistochemistry (C. Milstein and A. C. Cuello (1983) Nature 305: 537), and such.
Specific antibodies for a given antigen can be produced via genetic engineering, by obtaining the nucleotide sequences of heavy and light chain variable regions which determine the antigen specificity of antibodies (J. Xiang et al. (1990) Mol. Immunol. 27: 809; C. R. Bebbington et al. (1992) Bio/Technology 10:169). Methods for obtaining antigen-specific heavy chains and light chains include methods that utilize phages or phagemids using E. coli as the host (W. D. Huse et al. (1989) Science 246: 1275; J. McCafferty et al. (1990) Nature 348: 552; A. S. Kang et al. (1991) Proc. Natl. Acad. Sci. USA 88: 4363). In these methods, antibody libraries are constructed by generating Fabs, or by generating fusion proteins between a phage coat protein and Fab or single-strand Fv. Finally, antigenic affinity is examined to select antigen-specific antibodies and their genes from these antibody libraries.    [Patent Document 1] Published Japanese Translation of International Publication No. 2001-523971